Mammalian immune system plays a key role in controlling microbial infection in vivo. T cells play important roles in such immune responses. One class of T cells, the cytolytic T cells (CTL), function by lysing foreign and virally infected cells. The primary mechanism of CTL-mediated cytolysis involves directional release of cytoplasmic granule contents, by which CTL and NK cells initiate the lysis of the target cells.
The contents of the cytoplasmic granules include: a pore-forming protein (perforin), a family of serine proteases (granzymes), and a late T cell activation marker, granulysin. Granulysin is cytolytic against microbes and tumors. When attached to infected body cells, granulysis can create holes in the target cell membranes, leading to destruction of the cells. In addition, granulysin can induce apoptosis in the target cells and also has antimicrobial action. (Janeway, Charles (2005). Immunobiology: the immune system in health and disease (6th ed.). New York: Garland Science).
Human granulysin is expressed as proteins of two sizes (9 kDa and 15 kDa) derived from three unique transcripts. (see FIG. 1). Granulysin is saposin-like lipid binding protein. The crystal structure (Anderson et al. (2003) J Mol. Biol. 325(2):355-65) reveals a five-helix bundle with positive charges distributing in a ring around the molecule and one face without net positive charges (FIG. 2). In addition, granulysin is stabilized by two highly conserved intra-molecule disulfide bonds.
While immune responses are important in the defense against infections, unwanted immune responses may lead to disorders. Examples of disorders associated with unwanted immune responses include adverse drug reactions (ADRs), graft-versus-host diseases (GVHD), inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases, and T cell-derived cancers.
U.S. Pat. No. 7,718,378, issued to Chen et al., disclosed that granulysin is involved in the pathology of diseases associated with unwanted immunological responses or cytotoxic T cell mediated-disorders, such as SJS (Steven-Johnson syndrome), TEN (toxic epidermal necrolysis), and GVHD.
The pathogenesis of SJS/TEN is not fully understood. However, adverse drug reaction is a major cause of these conditions. In 2007, FDA issued an alert asking doctors to screen patients for human leukocyte antigen (HLA) allele, HLA-B*1502, before carbamazepine therapy because dangerous or even fatal skin reactions (SJS and TEN) can result from carbamazepine therapy with these patients. The manifestations of these serious life-threatening adverse drug reactions are believed to be immune-mediated since rechallenging with the same drug typically shortens the incubation period and results in more severe manifestations (Roujeau et al., Toxicology, 2005 Apr. 15; 209(2):123-9).
In addition, clinical, histopathological, immunocytological, and functional findings in SJS/TEN support the concept that SJS/TEN is a specific drug sensitivity reaction initiated by cytotoxic lymphocytes. Prior in vitro studies suggest that the drug presentation is MHC class I restricted, there is a clonal expansion of CD8+ CTLs, and these cells induce effector cytotoxic responses. The MHC-restricted presentation of a drug or its metabolites for T-cell activation is further supported by the recent findings of strong genetic association between HLA-B alleles and reaction to specific drugs. (Chung et al. Nature, 2004 Apr. 1; 428(6982):486.).
Cytotoxic T-cells are observed to infiltrate the skin lesions of SJS/TEN patients (Nassif et al., Allergy Clin. Immunol. 2004 November; 114(5): 1209-15). The T lymphocytes in the blister fluid and epidermis show a predominance of CD8+ phenotype (Nassif et al., J. Invest. Dermatol. 2002 April; 118(4):728-33). These observations point to a cutaneous recruitment of antigen-primed and cytotoxic T cells in the pathogenesis of SJS/TEN.
Granulysin was found to be a key molecule responsible for the unique clinical manifestation of SJS/TEN. Blister fluids from skin lesions of SJS/TEN patients exhibited cytolytic activity against B-cells and keratinocytes and contain granulysin as the most predominant cytotoxic protein. Furthermore, injection of granulysin into epidermis of mice induced massive skin cell death, mimicking the human pathology of SJS/TEN. Thus, granulysin is a key molecule responsible for the disseminated keratinocyte apoptosis and underlies the missing link of the pathogenic mechanism of SJS/TEN.
FIG. 3 shows a schematic illustrating a possible mechanism for the involvement of granulysin in adverse drug reactions (e.g., carbamazepine adverse reaction). According to this proposed mechanism, binding of the drug molecule (antigen) to MHC I on an antigen presenting cell (e.g., keratinocyte) leads to activation of CD8+ cytotoxic T cells. The drug-MHC I interaction is most significant when the allele is HLA-B*1502 and the drug is carbamazepine. Activation of the T cells leads to the production of granulysin, among other substances. Granulysin then triggers the apoptosis (and cytolysis) of keratinocyte.
In acute GVHD, granulysin was markedly increased in serum, and the levels of granulysin in serum correlated with the severity of GVHD. In addition, it was shown that allospecific T cells released granulysin in an allo-specific manner in vitro, and the granulysin release was correlated with allo-specific cytotoxic activity. These results indicate that granulysin plays an important role in GVHD. (Nagasawa et al. 2006, Am. J. Hematol. 81(5):340-8).
The above observations suggest that granulysin plays an important role in these unwanted immune response disorders. Therefore, granulysin is a useful target for diagnosis and therapy of such unwanted immune response disorders.